Project Summary/Abstract Placental insufficiency underlies many pregnancy disorders, including preeclampsia (PE), intrauterine growth restriction (IUGR) and recurrent pregnancy loss (RPL). Collectively, these disorders have costly, widespread, and sometimes long term, health consequences for the mother and/or child. Immune cell populations at the maternal-fetal interface, including uterine natural killer cells (uNKs) and uterine macrophages (uMacs), mediate angiogenesis and other key events in decidualization, placentation, and progression of pregnancy. Dysregulated inflammatory responses during decidualization or placentation have been linked to PE, IUGR, and RPL. Moreover, a growing number of births in the US are achieved using controlled ovarian stimulation (?superovulation?) protocols for in vitro fertilization (IVF), which is associated with dysregulated uterine immune cell function and increased risk of pregnancy complications due to placental dysfunction. The molecular mechanisms that regulate immune cell function at the maternal-fetal interface are poorly understood. Recent studies have highlighted critical roles for the IL-1 family cytokine, IL-33, in pregnancy, and aberrant maternal IL-33 signaling has been linked to RPL and PE in humans. Our preliminary data reveal numerous IL-33-expressing cells, including many IL-33+ uterine epithlelial cells, in the murine uterus at key stages of pregnancy. These IL-33+ cells are located in immune cell-rich regions heavily populated by uNKs and uMacs. Although IL-33 regulates key aspects of NK and Mac function in other tissues, its specific effects on uNKs and uMacs remains poorly understood. We hypothesize that IL-33 signaling supports decidualization and placentation by promoting uNK and uMac-mediated angiogenesis and tissue remodeling during pregnancy. Guided by this hypothesis, our proposed studies aim to: (1) Define the role of IL-33 signaling in pregnancy progression. We will utilize mice that lack IL-33 globally or in uterine epithelial cells, or mice in which IL-33 is neutralized at key stages of gestation, to define the role of IL-33 in decidualization, placental formation and placental function during pregnancy. (2) Determine the effects of IL-33 signaling on NK and Mac function at the maternal-fetal interface. We will use complementary in vitro approaches and in vivo studies in IL-33-deficient mice to determine how IL-33 signaling regulates the angiogenic and tissue remodeling activities of uNKs and uMacs during pregnancy. (3) Determine the impact of superovulation (SO) on uNK and uMac function. We will use mouse models of SO, in combination with studies on primary human endometrial samples from women undergoing SO for IVF, to investigate the impact of SO on IL-33-dependent and ?independent uNK and uMac effector functions.